Trehalose (glucose-α-1,1-α-glucose) is a non-reducing disaccharide with some very interesting properties (Higashiyama, 2002). It is present in a wide range of organisms (most notably yeast and plants), where it protects against environmental stresses such as heat, freezing and drought. It is also stable at a wide range of pH-values, has a mild sweet taste and is not cariogenic. These properties make it ideally suited for use in processed food. In the end, ingested trehalose is hydrolysed by intestinal trehalase (EC 3.2.1.28) and absorbed in the small intestine.
Trehalose is produced at an industrial scale from maltodextrins in a two-step process (Maruta et al, 1995). First, maltooligosyl trehalose synthase catalyses the conversion of the α-1,4-bond at the reducing end into an α-1,1-α-bond, after which the adjacent α-1,4-bond is hydrolysed by maltooligosyl trehalose trehalohydrolase to release trehalose. The industrial process was developed by Hayashibara and has allowed the production of trehalose at a very competitive price of a few /kg (EP0606753 and EP0628630). However, the process can not be used for the production of trehalose-analogues that contain other monosaccharides than glucose.
Analogues of trehalose could have some additional benefits with respect to their physicochemical and biological properties. Galactose-α-1,1-α-glucose (hereafter referred to as lactotrehalose), for example, is not hydrolysed by intestinal trehalase but functions as a competitive inhibitor (Kim et al., 2007). This means that this disaccharide does not contribute to the caloric content of food preparations and also lowers the metabolic conversion of trehalose. Unfortunately, trehalose-analogues are not yet produced at an industrial scale and have, therefore, not been studied very extensively.
The chemical synthesis of trehalose-analogues has been described several years ago (Youssef et al., 1995; Lee et al., 1976, Pratt et al., 2003). However, these procedures consist of multistep synthetic routes that have a low overall yield and generate a lot of waste. Alternatively, a glucosyltransferase has been described that catalyses the synthesis of lactotrehalose from UDP-glucose and galactose (Kim et al., 2007). Although very efficient, the need for an expensive nucleotide-activated donor prohibits the cost-efficient exploitation of this enzyme. Furthermore, other acceptors besides galactose can not be used, restricting its application to the synthesis of lactotrehalose.
Finally, the synthesis of trehalose-analogues has also been described for trehalose phosphorylase (Belocopitow et al., 1971; Aisaka et al., 2000; Chaen et al., 2001). This enzyme normally catalyses the degradation of trehalose into β-glucose-1-phosphate and D-glucose, but the reaction can also be run in the synthetic direction. In that case, a glucosyl phosphate is required as donor, which is much cheaper and more stable then a nucleotide-activated donor. Activity of trehalose phosphorylases on D-glucosamine, D-xylose, D-galactose, D-fucose, L-fucose and L-arabinose as acceptors has been reported, albeit at a reduced rate (Chaen et al. 1999). In addition, oligosaccharides with a reducing-end glucose residue are successful acceptors for the trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047 (Maruta et al., 2006). Chimeric phosphorylases have been created that combine parts of the kojibiose phosphorylase and of the trehalose phosphorylase from Thermoanaerobacter brockii (Yamamoto et al., 2006). Although the resulting enzymes have altered substrate specificities, they do not display activity towards new acceptors. U.S. Pat. No. 5,993,889 also discloses a trehalose phosphorylase obtainable from microorganisms of the genus Thermoanaerobium which can be used to synthesize trehalose-analogues and further indicates that functional equivalents of the enzyme can be obtained by treating the microorganisms with an appropriate mutagen. However, this document does not teach which specific mutation(s) will result in beneficial and useful properties of the enzyme and which specific mutation(s) will result in detrimental or useless properties of the enzymes.
There is thus still a need within industry to have access to alternative trehalose phosphorylases which are highly thermostable, have a broad acceptor specificity and have a high activity on the acceptors. Such trehalose phosphorylases are useful for the industrial production of trehalose-analogues. Trehalose phosphorylases having an optimized donor specificity are useful for the production of glycosyl phosphates.